The tumorsphere assay allows one to assess whether a single cell harbors the potential to both initiate and maintain tumors in the absence of cellular interaction and adhesion. Stem cells are capable of unlimited self-renewal ability, and unique subsets of cancer cells that acquire stem cell properties have the theoretical ability to form de novo tumors when grown under low-attachment conditions within minimal growth factor supplementation. Primary stem cells and progenitor cells from the breast can be enriched within mammospheres, a concept that is analogous to neural stem cell enrichment in neurospheres. Tumorigenic efficiency of a subset of cancer cells can be determined based on number of spheres that emerge from single cells. On the contrary, cancer cells that lack stem cell properties have limited sphere-forming potential due to telomere loss and cellular senescence.